Models Of Chromatin Structure Research Articles

Mixed secondary chromatin structure revealed by modeling radiation-induced DNA fragment length distribution

Spatial chromatin structure plays fundamental roles in many vital biological processes including DNA replication, transcription, damage and repair. However, the current understanding of the secondary structure of chromatin formed by local nucleosome-nucleosome interactions remains controversial, especially for the existence and conformation of 30 nm structure. Since chromatin structure influences the fragment length distribution (FLD) of ionizing radiation-induced DNA strand breaks, a 3D chromatin model fitting FLD patterns can help to distinguish different models of chromatin structure. Here, we developed a novel "30-C" model combining 30 nm chromatin structure models with Hi-C data, which measured the spatial contact frequency between different loci in the genome. We first reconstructed the 3D coordinates of the 25 kb bins from Hi-C heatmaps. Within the 25 kb bins, lower level chromatin structures supported by recent studies were filled. Simulated FLD patterns based on the 30-C model were compared to published FLD patterns induced by heavy ion radiation to validate the models. Importantly, the 30-C model predicted that the most probable chromatin fiber structure for human interphase fibroblasts in vivo was 45% zig-zag 30 nm fibers and 55% 10 nm fibers.

Integrating Structural and Functional Studies Leads to a New Model of β-Globin Activation That Suggests Distinct Initiation and Maintenance States

Abstract 349The human and mouse β-globin loci share a conserved structure in which the locus control region (LCR) and genes are flanked by three CTCF bound DNaseI hypersensitive sites (HSs); 3‘HS1 downstream, and 5‘HS5 and human and mouse orthologues HS-111 or HS-62 upstream. In mice HS-62 and 3‘HS1 delineate a DNase sensitive domain. During erythroid differentiation, high-level expression of the β-globin locus is associated with LCR-dependent re-localization of the locus from the nuclear periphery to the nucleoplasm, where it associates with foci of serine-phosphorylated PolII deemed transcription factories (TFs).To investigate the relationships among chromatin structure, nuclear localization and β-globin expression during human erythropoiesis, CD34 progenitor cells were differentiated and analyzed by ChIP-array, primary transcript FISH, immuno-FISH, and chromatin conformation capture, carbon copy (5C). Localization of the β-locus away from the nuclear periphery and to TFs, and detection of β-nascent transcripts are rare events at day 4 (proerythroblasts), whereas by day 15 (polychromatic erythroblasts), nearly all loci are centrally located, associated with TFs and actively expressing. Three megabase profiles of complementary active and repressive histone marks (H3 lysine 4 di-methylation (DiK4) and H3 lysine 27 tri-methylation) reveal that DiK4 is enriched in the LCR and adult genes in undifferentiated CD34 cells and nears maximal enrichment by day 4. Thus the chromatin landscape is set up prior to erythroid commitment and is increased at day 4, but shows little change with activation. These profiles also reveal a previously un-described 257kb domain spanning from HS-111 to +146 relative to the ε-gene cap, with CTCF bound at its boundaries.5C analysis reveals a high linkage frequency between the LCR and β-gene at day 4, prior to β-gene activation. Thus proximity may be necessary, but is not sufficient for high-level expression. In addition, the LCR and adult genes have frequent contact with surrounding regions, but interactions are sharply demarcated by HS-111 and +146, linking the above histone modification domain and 5C structure. The flanking regions HS-111, 3‘HS1 and +146 associate with the LCR and genes in an active chromatin hub (ACH)-like structure. By combining 5C with the Integrated Modeling Platform, a high-resolution three-dimensional (3D) model of chromatin structure was generated and revealed that the CTCF containing flanking regions, HS-111, 5’HS5, 3‘HS1 and +146 are in proximity and anchor loops of the intervening regions. The LCR and β-gene lie in close proximity (<100nm) within a tight chromatin globule. At day 15, 5C-interactions become more restricted. Throughout the 1Mb assayed there is a global decrease in linkages and, unlike the ACH model, associations of the flanking HSs with each other diminish. In contrast, the LCR and β-gene are more highly linked. While the distance between the LCR and β-gene remains under 100nm at day 15, most of the remaining 3D structure is less compact. One notable exception is with differentiation the LCR is in closer proximity to the β-gene 3‘enhancer. In addition, β-gene activation is associated with an increase in the contour length of the region, possibly correlating with DNaseI accessibility.In summary, our results reveal that acquisition of DiK4 precedes erythroid commitment. Enrichment of this active histone modification may occur in the nuclear periphery and is associated with a compact structure in which the flanking HSs, LCR and β-gene are in close proximity. Notably, close association of the LCR and β-gene precedes association with a TF and is not sufficient for expression. This suggests that if an ACH structure is important for function, its role may be limited to the initiation, but not the maintenance of gene expression. Alternatively, this compact structure may reflect the mafK-mediated recruitment of co-repressors to the LCR, as we demonstrated previously. The exchange of these repressors for activators could lead to the observed relocation from the periphery to TFs where high-level expression occurs and provides an explanation for the large change in expression that occurs despite a subtle change in the proximity of the LCR and β-gene. Preliminary studies on cell lines harboring intact, and LCR deficient chromosomes suggest the LCR may effect boundary element formation, domains of histone modifications and structure of the region. Disclosures:No relevant conflicts of interest to declare.

Quantitative Diameter Measurements of Chromatin and TMV Fibers in the Freeze-Dried State

Although there is general agreement that Inactive chromosome fibers consist of helically packed nucleosomes, the pattern of packing is still undetermined. Only one of the proposed models, the crossed-linker model, predicts a variable diameter dependent on the length of DNA between nucleosomes. Measurements of the fiber diameter of negatively-stained and frozen- hydrated- chromatin from Thyone sperm (87bp linker) and Necturus erythrocytes (48bp linker) have been previously reported from this laboratory. We now introduce a more reliable method of measuring the diameters of electron images of fibrous objects. The procedure uses a modified version of the computer program TOTAL, which takes a two-dimensional projection of the fiber density (represented by the micrograph itself) and projects it down the fiber axis onto one dimension. We illustrate this method using high contrast, in-focus STEM images of TMV and chromatin from Thyone and Necturus. The measured diameters are in quantitative agreement with the expected values for the crossed-linker model for chromatin structure

Cryo-electron microscopy of chromosome fibers is consistent with the crossed-linker model for chromatin structure

Although there is general agreement that inactive chromosome fibers consist of helically packed nucleosomes, the pattern of packing is still disputed. The nucleosome itself is composed of a highly conserved “core” comprised of 146bp of DNA and a histone octamer, and a variable “linker” comprised of 20-100bp of DNA bound to histone H1. The models of chromatin structure can be distinguished by their dependence upon linker length. The solenoid model for chromatin structure is a single helix with constant pitch (11 nm) and constant diameter (25-30nm). The twisted-ribbon model is a double helix with variable pitch (26-40nm) and constant diameter (30nm). The crossed-linker model is a double helix with conserved pitch (26-28nm) and variable diameter (26-40nm). Measurements of the diameter and apparent helical parameters of negatively-stained chromatin from Thyone sperm (87bp linker) and Necturus erythrocytes (48bp linker) are in agreement with a left-handed crossed-linker model, and in disagreement with the solenoid and twisted-ribbon models.

DNA wrapping in nucleosomes. The linking number problem re-examined.

Chromatin was assembled in vitro from relaxed closed circular DNA (SV40) and core histones at histone to DNA ratios of 0.2 to 0.3 (g/g) and incubated with topoisomerase I to relax supercoils in DNA regions not constrained by protein. Addition of histones H1 + H5 to the chromatin at an ionic strength of 0.1 M, in the presence of the solubilizing agent, polyglutamic acid, and topoisomerase I, increased the magnitude of the DNA linking number change, relative to protein-free DNA. No change in the linking number distribution occurred for relaxed protein-free DNA under these conditions. Control experiments indicated that the increase in the absolute value of the DNA linking number change in the chromatin could not be attributed to an increase in the number of nucleosomes per DNA molecule. These data suggest a solution to the linking number problem associated with models of chromatin structure.

Nucleosome Structure III: The Structure and Transcriptional Activity of the Chromatin Containing the Ovalbumin and Globin Genes in Chick Oviduct Nuclei

The demonstration that the bulk of eukaryotic chromatin is organized in a repeating subunit structure, the nucleosome (for references, see Kornberg 1977), raised the question as to whether all genes are packaged in a similar manner, since any model of chromatin structure should also account for the selective transcription of only a minor part of the genome. Studies based on micrococcal nuclease digestion of chromatin have suggested that actively transcribed ribosomal RNA genes (rRNA genes) are indeed in the bulk nucleosome structure (Reeves 1976; Reeves and Jones 1976; Mathis and Gorovsky 1976; Piper et al. 1976). However, in most of these studies the interpretation of the results is complicated by the fact that not all of the rRNA genes are necessarily active at a given time. The examination of specific structural gene sequences coding for mRNA provides a sharper focus on the question, although in this case, also, the possible...

Transcription of the nonrepeated fraction of "accessible" DNA in rat liver chromatin.

The relationship between structure and function in eukaryotic chromatin has been studied in rat liver cells. To elucidate the functional significance of "accessible" DNA, the transcription of this DNA (prepared by titration of liver chromatin with poly-D-lysine) has been examined by RNA-DNA hybridization. The maximum extent to which nuclear RNA will hybridize to the nonrepeated fraction of "accessible" DNA has been measured and compared to the extent that whole chromatin DNA will hybridize. The results show that "accessible" DNA has the same number of sequences complementary to nuclear RNA as does total DNA. In addition DNA-DNA reassociation experiments indicate that there is only a small difference between the total unique sequence populations of "accessible" and total DNA. These results indicate that nonrepeated "accessible" DNA is not preferentially transcribed in the cell as is predicted by some models of chromatin structure.

A model for chromatin structure.

A model for chromatin structure is presented. (a) Each of four histone species, H2A (IIbl or f2a2), H2B (IIb2 or f2b), H3 (III or f3) and H4 (IV or f2al) can form a parallel dimer. (b) These dimers can form two tetramers, (H2A)2(H2b)2 and (H3)2(H4)2. (C) These two tetramers bind a segment of DNA and condense it into a "C" segments. (d) The adjacent segments, termed extended or "E" segments, are bound by histone H1 (I or fl) for the major fraction of chromatin; the other "E" regions can be either bound by non-histone proteins or free of protein binding. (e) The binding of histones causes a structural distortion of the DNA which, depending upon the external conditions, may generate the formation of either an open structure with a heterogeneous and non-uniform supercoil or a compact structure with a string of beads. The model is supported by experimental data on histone-histone interaction, histone-DNA interaction and histone subunit-DNA interaction.

Structure of chromatin. DNA not complexed with protein.

Abstract Rat liver chromatin was titrated by poly-ᴅ-lysine of mol.wt. 140,000. Approximately 32% of the phosphate groups of the chromatin were found to react with the polycation. By submitting the chromatin-poly-ᴅ-lysine complex to pronase and DNase I digestion and by further submitting the obtained DNA-lysine complex to hydroxy-apatite-chromatography, "free" DNA was isolated. The mean S-value of this DNA was found to be 10 ± 0 .8 , corresponding to approximately 2100 nucleo­ tide pairs, its renaturation kinetics similar to total DNA. On the basis of these results and on the findings that histone-saturated DNA-histone recombinates are digested with DNase I up to 65% a model of chromatin structure was proposed, taking into account protein covered-and free DNA regions